ABSTRACT
Frizzled receptors are components of the Wnt signalling pathway, but how they activate the canonical Wnt/beta-catenin pathway is not clear. Here we use three distinct vertebrate frizzled receptors (Xfz3, Xfz4 and Xfz7) and describe whether and how their C-terminal cytoplasmic regions transduce the Wnt/beta-catenin signal. We show that Xfz3 activates this pathway in the absence of exogenous ligands, while Xfz4 and Xfz7 interact with Xwnt5A to activate this pathway. Analysis using chimeric receptors reveals that their C-terminal cytoplasmic regions are functionally equivalent in Wnt/beta-catenin signalling. Furthermore, a conserved motif (Lys-Thr-X-X-X-Trp) located two amino acids after the seventh transmembrane domain is required for activation of the Wnt/beta-catenin pathway and for membrane relocalization and phosphorylation of Dishevelled. Frizzled receptors with point mutations affecting either of the three conserved residues are defective in Wnt/beta-catenin signalling. These findings provide functional evidence supporting a role of this conserved motif in the modulation of Wnt signalling. They are consistent with the genetic features exhibited by Drosophila Dfz3 and Caenorhabditis elegans mom-5 in which the tryptophan is substituted by a tyrosine.
Subject(s)
Cytoplasm/chemistry , Cytoskeletal Proteins/metabolism , Proteins/chemistry , Proto-Oncogene Proteins/metabolism , Receptors, G-Protein-Coupled , Trans-Activators , Xenopus Proteins , Zebrafish Proteins , Adaptor Proteins, Signal Transducing , Amino Acid Sequence , Animals , Blotting, Western , Caenorhabditis elegans , Cell Membrane/metabolism , Cytoskeletal Proteins/chemistry , DNA, Complementary/metabolism , Dishevelled Proteins , Drosophila , Drosophila Proteins , Embryo, Nonmammalian/metabolism , Frizzled Receptors , In Situ Hybridization , Ligands , Molecular Sequence Data , Mutagenesis, Site-Directed , Phosphoproteins/genetics , Phosphoproteins/metabolism , Phosphorylation , Plasmids/metabolism , Point Mutation , Protein Structure, Tertiary , Proteins/genetics , Proteins/metabolism , Proto-Oncogene Proteins/chemistry , RNA, Messenger/metabolism , Receptors, Cell Surface/genetics , Receptors, Cell Surface/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Sequence Homology, Amino Acid , Signal Transduction , Time Factors , Wnt Proteins , Xenopus , beta CateninABSTRACT
Wnt signalling plays a crucial role in the control of morphogenetic movements. We describe the expression and functional analyses of frizzled 7 (Xfz7) during gastrulation in Xenopus. Low levels of Xfz7 transcripts are expressed maternally during cleavage stages; its zygotic expression strongly increases at the beginning of gastrulation and is predominantly localized to the presumptive neuroectoderm and deep cells of the involuting mesoderm. Overexpression of Xfz7 in the dorsal equatorial region affects the movements of convergent extension and delays mesodermal involution. It alters the correct localization, but not the expression, of mesodermal and neural markers. These effects can be rescued by extra-Xfz7, which is a secreted form of the receptor that also weakly inhibits convergent extension when overexpressed. This suggests that the wild-type and truncated receptors have opposing effects when coexpressed and that overexpression of Xfz7 causes an increased signalling activity. Consistent with this, Xfz7 biochemically and functionally interacts with Xwnt11. In addition, Dishevelled, but not (&bgr;)-catenin, synergizes with Xfz7 to affect convergent extension. Furthermore, overexpression of Xfz7 and Xwnt11 also affects convergent extension in activin-treated animal caps, and this can be efficiently reversed by coexpression of Cdc42(T17N), a dominant negative mutant of the small GTPase Cdc42 known as a key regulator of actin cytoskeleton. Conversely, Cdc42(G12V), a constitutively active mutant, rescues the effects of extra-Xfz7 on convergent extension in a dose-dependent manner. That both gain-of-function and loss-of-function of both frizzled and dishevelled produce the same phenotype has been well described in Drosophila tissue polarity. Therefore, our results suggest an endogenous role of Xfz7 in the regulation of convergent extension during gastrulation.
Subject(s)
Gastrula/physiology , Receptors, Cell Surface/genetics , Receptors, G-Protein-Coupled , Trans-Activators , Xenopus Proteins , Xenopus laevis/embryology , Adaptor Proteins, Signal Transducing , Animals , Biomarkers , Body Patterning/genetics , Cytoskeletal Proteins/genetics , Cytoskeletal Proteins/metabolism , Dishevelled Proteins , Drosophila Proteins , Ectoderm/cytology , Ectoderm/physiology , Embryo, Nonmammalian , Gene Expression Regulation, Developmental , Genes, Dominant , Glycoproteins/genetics , Glycoproteins/metabolism , Mesoderm/cytology , Mesoderm/physiology , Mutation , Phosphoproteins/genetics , Phosphoproteins/metabolism , Receptors, Cell Surface/metabolism , Signal Transduction , Wnt Proteins , Xenopus laevis/genetics , beta Catenin , cdc42 GTP-Binding Protein/genetics , cdc42 GTP-Binding Protein/metabolismABSTRACT
We have investigated the adenylyl cyclase (AC) activity and gene expression in retinoic acid (RA)-primed murine P19 teratocarcinoma cells, which recapitulate in vitro the first stages of neuroectodermal formation. Here we show that the P19 stem cells possess a basal Ca2+/CaM-stimulated AC activity, which increases about 10-fold after RA induction. The rise of AC activity is associated with a stage-specific up-regulation of AC2, AC5 and AC8 mRNAs and a down-regulation of AC3 mRNA. P19 cells provide a powerful model to investigate the role and specific regulation of AC isoforms during neuronal differentiation.
